There are no chemotherapeutic treatments capable of extending life past a few months for patients with hormone refractory prostate cancer. Prostate-specific conditionally replicating adenoviruses (CRADs) offer a safe, auxiliary platform that has previously shown promise in early phase clinical trials for local disease. However, the natural bio-distribution of adenovirus and the inability to image therapeutic effect have limited their application in metastatic disease. Our long term goal is therefore to improve existing prostate specific CRADs by (1) re-targeting viral transduction through alternative receptors and (2) generating a Positron Emission Tomography (PET) imaging reporter that is specific for viral replication. We have designed a novel adenoviral vector system which applies specialized cre/lox mediated gene cassette exchange in the fiber gene locus to help achieve these goals. We hypothesize that adenovirus modified to ablate natural infection pathways (de-targeting) with redirection for infection through prostate cell surface markers (re-targeting) will improve biodistribution in animal models. Further, an innovative adenoviral library selection strategy is proposed to overcome potential interferences associated with viral re-targeting. The same vector system offers means to insert temporally regulated imaging reporter genes specific to adenoviral replication. Fiber-modified vectors will therefore be generated with this novel system to achieve the following specific aims: 1) To generate and characterize multiple adenovirus vectors displaying PSMA and other prostate binding-peptides in the fiber Hl-loop in the background of Coxsackie and Adenovirus Receptor (CAR), integrin, and heparin sulfate binding mutations or CAR and blood factor binding mutations. Transductional specificity of these vectors will be tested on multiple cell lines in vitro, followed by prostate cancer xenograft models in vivo. 2) To generate a low diversity adenovirus peptide display library based on a single prostate targeting peptide, flanked by two random sequence three amino acid cassettes, and to use viral infection and production yield to identify the viruses that best infect cells via PSMA. 3) To generate and characterize a PET imaging reporter system capable of specifically imaging adenoviral replication by linking expression of the optimized Herpes Symplex Virus Thymidine Kinase (HSV1-sr39TK) reporter gene to the major late fiber gene through an internal ribosome entry site.